Engineering individually addressable cellular spheroids using aqueous two-phase systems

ABSTRACT

Provided are multi-phase systems that may be used to prepare a three-dimension aggregate of cells referred to as a cellular spheroid. The multi-phase system includes a droplet of an aqueous polymer phase within an immersion aqueous polymer phase. The droplet of the droplet aqueous polymer phase contains a three-dimensional aggregate of cells (cellular spheroid). Types of cells that may be used in the multi-phases system include stem cells and cancer cells. The cellular spheroids with the multi-phase system may be used to monitor cell growth in three-dimensional systems, or screen drugs in a three-dimension aggregate of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/064,886, filed Oct. 28, 2013, which claims priority from U.S.Provisional Patent Application No. 61/718,775 filed on Oct. 26, 2012,all incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to three dimensional cell aggregates or“cellular spheroids.” More particularly the present invention provides acellular spheroid in an aqueous two-phase system, methods of preparing acellular spheroid in an aqueous two-phase system, and methods ofscreening a drug with a cellular spheroid in an aqueous two-phasesystem.

BACKGROUND OF THE INVENTION

Conventional two-dimensional cultures of cancer cells are widely usedfor evaluating potential anti-cancer drugs. However, growing evidenceshows that cancer cells respond differently to anticancer drugs in atwo-dimensional monolayer culture than they would in vivo, where cellsreside in a three-dimensional environment. To more accurately model theeffects of potential drugs in vivo a three dimensional aggregate ofcells, or cellular spheroid, is desired. Presently, the hanging dropmethod is used to produce cancer cell spheroids for anti-cancer drugscreening. The hanging drop method, which produces spheroids bysuspending cells in droplets of medium, suffers from severalshortcomings. For instance, surface tension limits the maximum size of adrop prepared by the hanging drop method, also, due to the small size ofthe suspended drops, evaporation is a large concern. As the water withinthe drop evaporates, the concentration of soluble components such asproteins and salts in the medium increases, subjecting the cells to achanging osmotic pressure, thus compromising their normal morphology andfunction. The media also needs to be refreshed daily to avoid the buildup of cell waste and because of the small volume of media available tothe cells. The exchange of media is generally done by hand, using apipette, increasing the likelihood of incorporating errors such asaspirating out spheroids from hanging drops or introducing shear stressto cells due to manual pipetting. In addition, treating spheroids withexact drug concentrations is a challenge due to the presence of existingmedia. The hanging drop method is also sensitive to physical movementsthat can result in the detachment of drops and the spheroids within fromthe surface from which the drops hang.

Presently, a need exists for methods of screening drug compounds inthree-dimensional cultures that do not suffer from the above problems,and thus enable reliable, high throughput screening of potential drugcompounds.

SUMMARY OF THE INVENTION

An embodiment of the present invention provides an aqueous two-phasesystem for cell aggregates comprising: an immersion aqueous polymerphase; and, within the immersion aqueous polymer phase, a droplet of adroplet aqueous polymer phase containing a three-dimensional aggregateof cells.

Another embodiment provides a method of producing a cellular spheroidcomprising the steps of: providing a well containing a immersion aqueouspolymer phase; inserting a droplet of a droplet aqueous polymer phase;inserting cells into the droplet aqueous polymer phase; and allowing thecells to self assemble into a spheroid.

Another embodiment provides a method of screening potential drugscomprising: providing an aqueous two-phase system for the production ofcell spheroids comprising a immersion aqueous polymer phase, and withinthe immersion aqueous polymer phase, a droplet of a droplet aqueouspolymer phase containing a cellular spheroid; administering a potentialdrug; and monitoring the cellular spheroid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic of one or more embodiments of an aqueoustwo-phase system made from culture media and polyethylene glycol anddextran as two phase-forming polymers. Dextran phase is denser and canform a drop within the PEG phase sitting on the surface, if a smallvolume of the DEX phase is dispensed into the PEG phase.

FIG. 1B provides an actual image of one or more embodiments of amulti-phase system where a DEX drop is formed within the immersion PEGphase.

FIG. 2A provides a schematic of a dextran phase drop containing cellsimmersed in the immersion polyethylene glycol phase.

FIG. 2B provides a schematic of cells within the dextran drop aggregateand form a spheroid.

FIG. 3 provides a spheroid of A431.H9 skin cancer cells formed insidethe dextran drop immersed in the polyethylene glycol phase. Theperiphery of the dextran drop is visible.

FIG. 4A shows a distribution of diameter of 0.3 microliters DEX dropscontaining cells after 24 hrs and 48 hrs of incubation.

FIG. 4B shows spheroid diameter distribution after 24 hrs and 48 hrswith average values of 333±28 μm and 349±28 μm, respectively, for DEXdrops of 0.3 microliters containing 10,000 skin cancer A431.H9 cells.

FIG. 5 shows circularity of spheroids and circularity of DEX drops of0.3 microliters volume after 24 hrs and 72 hrs of incubation. A celldensity of 10,000 was used.

FIG. 6 shows spheroids growth curves for four cell densities over 7 daysof incubation.

FIG. 7A provides the resulting spheroids from a 5,000 cells/0.3 μldensity using MDA-MB-157 cells.

FIG. 7B provides the resulting spheroids from a 10,000 cells/0.3 μldensitys using MDA-MB-157 cells.

FIG. 7C provides the resulting spheroids from a 5,000 cells/0.3 μldensity using MDA-MB-157 cells.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one or more embodiments, an aqueous multi-phase system is providedcomprising a first aqueous polymer phase which may be referred to as animmersion aqueous polymer phase or immersion phase, and within theimmersion aqueous polymer phase, a droplet of a second aqueous polymerphase, which may be referred to as a droplet aqueous polymer phase ordroplet phase. Cells may be placed in the droplet of the droplet aqueouspolymer phase to produce a three-dimensional aggregate of cells, orcellular spheroid.

The aqueous multi-phase system includes at least two aqueous polymerphases that are immiscible with each other. The immiscible aqueouspolymer phases, if mixed, form distinct phases that can separate. Theaqueous polymer phases may be immiscible because a polymer in an aqueouspolymer phase has property that repels the polymer in another aqueouspolymer phase. For instance, an immersion aqueous polymer phase may byhydrophobic and repel the polymers in a hydrophilic droplet aqueouspolymer to form separate phases.

In one or more embodiments, the immersion aqueous polymer phase is lessdense than the second aqueous polymer phase. In these embodiments, adroplet polymer phase will reside at the bottom of the immersion aqueouspolymer phase. In other embodiments, the immersion aqueous polymer phaseis more dense than the droplet aqueous polymer phase. In theseembodiments, the droplet polymer phase will reside at the top of theimmersion aqueous polymer phase. In these or other embodiments, themulti-phase system may be a three-phase system, where the dropletaqueous polymer phase is less dense than the immersion aqueous polymerphase, but more dense than a third aqueous polymer phase. In theseembodiments, the droplet polymer phase will reside at the interface ofwhere the immersion and third aqueous polymer phase meet.

In one or more embodiments, the total volume of the multi-phase polymersystem is large enough to avoid the deleterious effects of solventevaporation. The solvent evaporation, and its effects, may be limited byusing larger total volumes of the multi-phase polymer system. Thisallows the multi-phase polymer system to be used for long term cellcultures. Herein, a long term cell culture is one in which cells can becultured for greater than one week. Further, because the multi-phasepolymer system does not require the use of a hanging drop, the totalvolume is not limited to the volume that can be suspended. Thus, themaximum volume of multi-phase polymer system is limited by pragmaticconcerns. In these or other embodiments, a long term culture may be 6 to8 weeks. In one or more embodiments, the multi-phase polymer system hasa total volume of greater than 10 μL, in other embodiments, greater than25 μL, and in still other embodiments, greater than 50 μL.

The use of the term “droplet” herein refers to the size and shape of thedroplet phase, and does not necessarily reflect how the droplet phase isformed or introduced into the first phase. In one or more embodiments,the droplet of the droplet aqueous polymer phase is essentiallyspherical. A droplet that is essentially spherical refers to a dropletshape that is spherical or close to spherical and has a round surfacewithout any flat spots or with only minimal flat spots.

The droplet may be characterized by the volume of the droplet. In one ormore embodiments the droplet has a volume of less than 1000 nL, in otherembodiments, less than 500 nL, and in still other embodiments, less than300 nL. In these or other embodiments, the droplet has a volume ofgreater than 20 nL, in other embodiments, greater than 50 nL, and instill other embodiments, greater than 100 nL. In certain embodiments thedroplet has a volume of about 20 nL to about 1000 nL, in otherembodiments, of about 50 nL to about 500 nL, and in still otherembodiments, of 100 nL to about 300 nL.

The droplet may also be characterized by its volume percentage of thetotal aqueous multi-phase system. In one or more embodiments, thedroplet makes up less than 0.02%, in other embodiments, less than 0.01%and in still other embodiments, less than 0.005% of the total aqueousmulti-phase system. In these or other embodiments, the droplet has apercent volume of greater than 0.0001%, in other embodiments, greaterthan 0.0002%, and in still other embodiments, greater than 0.0004% thetotal aqueous multi-phase system. In certain embodiments, the droplethas a percent volume of about 0.02% to about 0.0001%, in otherembodiments, of about 0.01% to about 0.0002%, and in still otherembodiments, of about 0.005% to about 0.0004% the total aqueousmulti-phase system.

Each aqueous polymer phase comprises a polymer dissolved in water. Theamount of polymer dissolved in water may be described by the percentweight of polymer in total weight of the aqueous polymer phase. Itshould be noted that, with smaller polymers are used, typically a largerpercent weight of polymer is needed to properly form separate aqueouspolymer phases with another aqueous polymer. In one or more embodiments,the amount of polymer is less than 20%, in other embodiments, less than18%, and in still other embodiments, less than 15% by weight of theaqueous polymer phase. In these or other embodiments, the amount ofpolymer is greater than 5%, in other embodiments, greater than 6%, andin still other embodiments, greater than 8% by weight of the aqueouspolymer phase. In certain embodiments, the amount of polymer is about 5%by weight to about 20% by weight in other embodiments, of about 6% byweight to about 18% by weight, and in still other embodiments, of about8% by weight to about 15% by weight of the aqueous polymer phase.

The polymer of the first aqueous polymer phase and the polymer of thesecond aqueous polymer phase in a two-phase polymer system may bereferred to as a polymer pair. While other polymer pairs may be used toprepare a two-phase polymer system, for ease of illustration, thepolymer pair of polyethylene glycol “PEG” and dextran “DEX” will bediscussed in further detail to describe the aqueous polymer phases.

A two-phase polymer system may be prepared with either an aqueouspolymer phase of dextran, which may be referred to as a dextran phase,or polyethylene glycol, which may be referred to as a polyethyleneglycol phase as the immersion aqueous polymer phase. Because the dextranphase is denser than the polyethylene glycol phase, the dextran phasewill reside at the bottom of the two-phase as seen in FIG. 1. A third,aqueous polymer phase may be added to produce a three-phase system. Ifthe third phase is denser than both the polyethylene glycol phase andthe dextran phase the dextran phase will reside as a droplet between thetwo phases.

A two-phase system may also be prepared from a dextran phase and apolyethylene glycol phase, where dextran is the immersion aqueouspolymer phase. In this two-phase system, polyethylene glycol phase isthe droplet aqueous polymer phase, and will reside at the top of thetwo-phase system. Again, a third, aqueous polymer phase may be added toproduce a three-phase system. If the third phase is less dense than boththe polyethylene glycol phase and the dextran phase the polyethyleneglycol phase will reside as a droplet between the two phases.

Suitable dextran polymers for use as a dextran phase may becharacterized by their molecular weight. In one or more embodiments, themolecular weight of the dextran polymer is less than 2,000,000 g/mol, inother embodiments, less than 1,000,000 g/mol, and in still otherembodiments, less than 500,000 g/mol the total aqueous multi-phasesystem. In these or other embodiments, the molecular weight of thedextran polymer is greater than 1000 g/mol, in other embodiments,greater than 10,000 g/mol, and in still other embodiments, greater than40,000 g/mol the total aqueous multi-phase system. In certainembodiments, the molecular weight of the dextran polymer is about 1000g/mol to about 2,000,000 g/mol, in other embodiments, of about 10,000g/mol to about 1,000,000 g/mol, and in still other embodiments, of about40,000 g/mol to about 500,000 g/mol the total aqueous multi-phasesystem.

Suitable polyethylene glycol polymers for use as a polyethylene glycolphase may be characterized by their molecular weight. In one or moreembodiments, the molecular weight of the polyethylene glycol polymer isless than 35,000 g/mol, in other embodiments, less than 20,000 g/mol,and in still other embodiments, less than 13,000 g/mol the total aqueousmulti-phase system. In these or other embodiments, the molecular weightof the polyethylene glycol polymer is greater than 100 g/mol, in otherembodiments, greater than 1,000 g/mol, and in still other embodiments,greater than 4,000 g/mol the total aqueous multi-phase system. Incertain embodiments, the molecular weight of the polyethylene glycolpolymer is about 100 g/mol to about 35,000 g/mol, in other embodiments,of about 1,000 g/mol to about 20,000 g/mol, and in still otherembodiments, of about 4,000 g/mol to about 13,000 g/mol the totalaqueous multi-phase system.

Again, other polymer pairs may be used to prepare a two-phase polymersystem as long as the polymers meet the minimum concentration at aspecific molecular weight to separate into different phases. Examples ofpolymer pairs other than polyethylene glycol and dextran suitable forpreparing a two phase polymer system include, but are not limited to,any two polymers selected from the group consisting of polyethyleneglycol, polyacrylamide, ficoll, poly(methyl methacrylate), andhydroxypropyl.

Any type of cells that exist in a three-dimensional environment in vivomay be used in the multi-phase system to produce a cellular spheriod. Inone or more embodiments, more than one type of cell may be used in themulti-phase system to provide a multicellular three-dimensionalaggregate that contains at least two different types of cells.

In one or more embodiments, the cells in the multi-phase system may bestem cells. An example of a type of stem cell includes, but is notlimited to, embryonic stem cells. The multi-phase system with embryonicstem cells may be used for the study of normal embryonic development.

In one or more embodiments, the cells in the multi-phase system may becancer cells. The multi-phase system with cancer cells may be used tomodel tumor growth. The multi-phase system also provides an idealenvironment for in vitro testing of potential drugs to inhibit tumorgrowth or eliminate cancer. Examples of cancer cells include but are notlimited to breast cancer, prostate cancer, liver cancer, lung cancer,ovarian cancer, and sarcomas cancer.

In one or more embodiments, cancer cells may be obtained from a cancerpatient. In these or other embodiments, cancer cells from a cancerpatient are retrieved during surgery and maintained as tumor xenograftin mouse. The cells may then be placed in a multi-phase polymer systemto produce cellular spheroids.

In one or more embodiments, the multiphase system includes cell media.Any aqueous polymer phase within the multi-phase system may include cellmedia. Cell media may be included in the first polymer phase, the secondpolymer phase, any additional polymer phases, or any combinationthereof. Those skilled in the art will appreciate the particular cellmedia required for the type of cells and the cellular spheroids desired.Examples of cell media include, but are not limited to, Dulbecco'smodified eagle medium and alpha minimum essential medium supplementedwith serum and other necessary ingredients.

In one or more embodiments, the multiphase system includes anextracellular matrix. Any aqueous polymer phase within the multi-phasesystem may include an extracellular matrix material. An extracellularmatrix may be included in the first polymer phase, the second polymerphase, any additional polymer phases, or any combination thereof. Theextracellular matrix may be used to provide structural support forcells, regulating intercellular communication, or regulatingcommunication between the cells and the extracellular matrix itself.Examples of extracellular matrix material include, but are not limitedto, collagen, laminin, fibronectin, matrigel, elastin, and combinationsthereof.

In one or more embodiments, the multi-phase system can be contained inwhat will be referred to as a well. Broadly, a well simply provides aholding volume for holding the multi-phase system. The well may bedepression formed with suitable structure and a bottom surface that isround, flat, or conical. In other embodiments, the multi-phase systemcan be may be supported on a flat bottom surface and the surface tensionof the phases will allow the immersion phase of the multi-phase systemto form a bead or hemispherical shape. Regardless, the multi-phasesystem is supported by or contained in depression that includes a bottomsurface. In one or more embodiments, the droplet has minimal contactswith the bottom surface. Minimal contacts with the bottom surface refersto the droplet's ability to form a substantially spherical shapeproviding a limited contact area between the bottom surface and thedroplet. Ideally, a droplet should have a contact angle as close aspossible to 180°.

In one or more embodiments, the bottom surface is non-adherent to cells.Typically, a surface that is non-adherent to cells has been treated witha coating that greatly reduces the binding of attachment proteinsproduced by the cells, thus minimizing cell attachment. Examples ofcommercially available plates, dishes, or flasks with a bottom surfacesthat are non-adherent to cells include ultra-low attachment platesavailable from Corning.

In one or more embodiments, a plurality of multi-phase systems may beprepared on the same apparatus, which may be referred to as a plate. Inthese or other embodiments, when the multiphase system is contained in awell, a plate may have a number of wells. Examples of plates with anumber of wells includes, but is not limited to, 6 well plates, 24 wellplates, 96 well plates, and 384 well plates. In other embodiments, wherethe multi-phase system is on a flat bottom surface, the flat bottomsurface may be a plate with any number of multi-phase systems.

An aqueous multi-phase system may be used to produce cellular spheroidsfrom cells. In one or more embodiments, a cellular spheroid may beprepared by providing a first aqueous polymer phase; inserting a dropletof a second aqueous polymer phase; inserting cells into the secondaqueous polymer phase; and allowing the cells to self assemble into aspheroid.

In one or more embodiments, the droplet of the second aqueous polymerphase may be inserted into the first aqueous polymer phase by pipettingsecond aqueous polymer phase into the first aqueous polymer phase. Inone or more embodiments, the second phase and cells are premixed priorto being inserted into the first aqueous polymer phase. In theseembodiments, cells and the second aqueous polymer phase are insertedinto the first polymer phase together.

In one or more embodiments, a cell culture media may be included bypremixing an aqueous polymer phase with the extracellular matrix. Inthese or other embodiments, it may be advantageous to refresh the cellculture media. Refreshing of cell culture media is typically performedby removing a portion of the cell culture media within the two phasesystem and replacing it with new cell culture media. Additional culturemedia may be added to replace any volume of liquid lost to evaporation.

In one or more embodiments, an extracellular matrix may be included bypremixing an aqueous polymer phase with the extracellular matrix.

While cellular spheroids may be produced from cells in an aqueousmulti-phase system prepared by hand, higher precision may be obtainedwhen a robotic apparatus is employed. In one or more embodiments, atleast one step is performed by a robotic apparatus. A robotic apparatusincludes the use of a liquid handler unit.

Steps performed by a robotic apparatus may be automated and performedwithout the assistance of humans. For instance, a liquid handling unitmay be programmed to store a plate, retrieve a plate, and refresh thecell culture media at regular intervals such as every three days. Othersteps may be included such as a photographing step, where a handlingunit may also be programmed to retrieve a plate and photograph the cellsin the aqueous multi-phase at regular intervals to chart spheroidprogression.

Advantageously, the used of a robotic apparatus allows for cellspheroids to be produced and analyzed in a high-throughput fashion. Inthese or other embodiments, a plurality of multi-phase systems asdescribed above may be used.

An aqueous multi-phase system may be used in a method to screenpotential drugs comprising: providing an aqueous two-phase system forthe production of cell spheroids comprising an immersion aqueous polymerphase, and within the immersion aqueous polymer phase, a droplet of adroplet aqueous polymer phase containing a cellular spheroid;administering a potential drug; and monitoring the cellular spheroid.

The term “potential drug” refers to a compound or compounds to be to beanalyzed for activity on a cell or cellular spheroid. A potential drugmay be administered to the cellular spheroid by introducing thepotential drug with the cell media during the creation of themulti-phase polymer system or during a cell media refresh. The potentialdrug may be administered to the cellular spheroid without cell media ina neat fashion or diluted in a solution.

The cells may be monitored by periodic viewing, and photographing. Inone or more embodiment, a stain may be applied to the cells of thecellular spheroid. The stain may be used to assist the visualdetermination of cell life. A stain useful for determining if the cellsare still alive is calcein AM. Other stains may be used to determine ifcells are no longer alive, such as ethidium homodimer-1. Assays may alsobe performed to monitor the effects of potential drugs on the cellularspheroids. Potential assays include, but are not limited to, Alamarblue, Presto blue, and MTT ((3-(4 5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide).

In one or more embodiments, where a plurality of multiphase systems areused to screen potential drugs, a potential drug may be administered indifferent concentration. In one or more embodiments, where a pluralityof multiphase systems are used to screen potential drugs, a differentpotential drug may be administered to each multiphase system or groupsof multiphase systems within a plurality of multiphase systems. In otherembodiments, the same drug my be administered to a plurality ofmultiphase systems or a plurality of groups of multiphase systems eachwith a different type of cell.

In order to demonstrate the practice of the present invention, thefollowing examples have been prepared and tested. The examples shouldnot, however, be viewed as limiting the scope of the invention. Theclaims will serve to define the invention.

EXAMPLES

The Example 1 was prepared using a two-component cell culture media.Component 1 contains 5% (w/w) polyethylene glycol (PEG, Mw: 35,000) andcomponent 2 contains 12.8% (w/w) Dextran (DEX, Mw: 500,000). When mixed,these two components segregate and form two immiscible phases that canbe separated (FIG. 1). First, the PEG phase is loaded into wells of awell plate (destination plate) and this plate is placed on the worksurface of a robotic liquid handler. Then cells are harvested from aculture dish, mixed with the DEX phase at a 1:1 volumetric ratio toreduce DEX concentration to 6.4% (w/w), and loaded into a second plate(source plate). This plate is also placed on the work surface of theliquid handler. Pipette tips are mounted onto the dispense head of theliquid handler and are immersed into the source plate to load a definedvolume (˜0.3 microliters) of the cell suspension in DEX phase. Afterretraction from the source plate, pipette tips are lowered into thedestination plate and dispense their content. Dispensed solution forms asingle droplet containing cells in each well and the droplet remainsimmiscible from its surrounding PEG phase (FIG. 2a ). Cells aggregateand form an individual spheroid in each well (FIG. 2b and FIG. 3).

TABLE 1 Aqueous Two-Phase Systems that Produced Spheroids Example TopPhase Bottom Phase 2  5% PEG 35K 5% DEX 500K 3  5% PEG 35K 10% DEX 500K4 15% PEG 35K 10% PAM 10K, 50 wt. % in H₂O 5 10% PEG 8K 10% DEX 500K 610% PEG 8K 5% DEX 500K 7 20% PEG 8K 5% DEX 500K

Table 1 provides a list of other aqueous two-phase systems that producedspheroids. In table 1, the PAM denotes polyacrylamide.

The consistency of spheroid size is critical for drug testing anddepends on the ability to dispense a well-defined volume of acell-containing DEX drop in each well. To ensure consistency ofDEX-drops, 80 drops of 0.3 microliters containing 10,000 skin cancercells (A431.H9) are dispensed into PEG solution in individual wells of a96-well plate and imaged after 24 hrs and 48 hrs. Captured images areanalyzed by image processing program, available as ImageJ from theNational Institute of Health, for drop and spheroid diameters. FIG. 4A,shows the distribution of drop diameter and gives the average andstandard deviations. After 24 and 48 hrs of incubation, the standarddeviation of the drop diameter is less than 8%, demonstrating theconsistency of our protocol. FIG. 4B, presents the diameter of spheroidsthat distributes with averages of 333 μm and 349 μm after 24 and 48 hrs,respectively. A standard deviation of smaller than 8.4% in addition to,a linear cumulative curve (inset of FIG. 4B) confirms the normaldistribution of spheroids diameter and the consistency of our spheroidformation technique.

To measure the circularity of forming spheroids, the aspect ratiodefined as largest versus smallest diameter of a spheroid is comparedversus that of DEX drops after 24 hrs and 72 hrs (FIG. 5). Moreover, toensure viability of spheroids, generated spheroids are stained with cellviability indicating fluorescent dyes. Live cells are stained green witha cell-permeable green fluorescent dye, calcein AM and dead cells arestained with a red fluorescent dye, ethidium homodimer-1. The cellstaining showed that cells are mainly stained green indicating theviability of cells. Less green color in the core of the spheroidrepresents reproducing a physiologic aspect of tumor spheroids that islimitation of diffusion of chemicals including drug compounds to thecore of spheroids.

Another important indication of viability of spheroids generated usingATPS microtechnology is the growth of spheroids during incubation. Overtime spheroids grow in size, resembling growth of tumors in cancerpatients (FIG. 6). To evaluate the growth patterns of spheroids formedusing the ATPS technology, four different cell densities of 2,500,3,500, 5,000 and 7,500 are studied. Spheroids are produced using 0.2microliters drops of 6.4% DEX residing within a 5% PEG solution asdescribed above. Spheroids are imaged every other day and 40 μl of freshmedia is robotically added to each well after imaging. FIG. 6 shows thegrowth curves of spheroids for all four cell densities. The diameter ofspheroids increases consistently for all densities demonstrating theviability of cells. In addition, spheroids diameter increases with anapproximate linear trend for all densities. The percentile growthdeclines slightly for higher cell densities. For example, the 7,500 celldensity spheroids show 35% change in diameter from the 1st to the 7thday whereas the 2,500 density spheroids show 52% change during the sametime period. However, spheroids with higher cell densities show moresignificant growth ratio (slope of growth curve) during the entireexperiment. It can be seen that the slope of growth line for the 2,500cell density spheroids is 12 μm/day whereas for spheroids with 2,500cell density, the slope is 17 μm/day. This is more prominent after the3rd day of incubation.

In addition, we show the versatility of this technology for generatingspheroids of different cancer cells by studying a metastatic breastcancer cell MDA-MB-157. FIG. 7A-C shows spheroid formation by thesecells at different cell densities. Finally, we demonstrate the potentialof this tumor spheroid technology for drug screening by treating bothA431.H9 and MDA-MB-157 cells with different concentrations of a standardanti-cancer drug, cisplatin up to 333.3 μM for A431.H9 cells and up to200 μM for MDA-MB-157 cells (Table 2). After incubation for four days, aviability reagent is added to each well and the viability of spheroidsis determined through an enzymatic assay available as PRESTO BLUE fromLife Technologies Co.

TABLE 2 Percent viabilities and standard errors for MDA-MB 157 spheroidstreated with cisplatin Cisplatin Percent Standard ConcentrationViability Error  25 μM 54.86% 12.81%  50 μM 34.31% 12.21% 100 μM 30.30%9.26% 150 μM 24.60% 4.73% 200 μM 18.81% 4.42%

Table 2 represents the viability of treated versus non-treated (control)spheroids against drug concentration produced. The drug, diluted withculture media, was renewed once by the addition of 50 μL after two daysof incubation. As displayed in the Table 1, the percent viabilityresults follow a gradual decrease as the cisplatin concentrationincreases. These results indicate the reliability of this assaytechnology for high throughput drug testing against cancer cellspheroids.

Various modifications and alterations that do not depart from the scopeand spirit of this invention will become apparent to those skilled inthe art. This invention is not to be duly limited to the illustrativeembodiments set forth herein.

What is claimed is:
 1. An aqueous two-phase system for cell aggregatescomprising: an immersion aqueous polymer phase; and within the immersionaqueous polymer phase, a droplet of a droplet aqueous polymer phasecontaining a three-dimensional aggregate of cells.
 2. The aqueoustwo-phase system of claim 1, where the three-dimensional aggregate ofcells is a cellular spheroid.
 3. The aqueous two-phase system of claim2, where the cellular spheroid is a multicellular spheroid containing atleast two different types of cells.
 4. The aqueous two-phase system ofclaim 1, where the immersion aqueous polymer phase is immiscible withthe droplet aqueous polymer phase.
 5. The aqueous two-phase system ofclaim 1, where the droplet aqueous polymer phase is denser than theimmersion aqueous polymer phase.
 6. The aqueous two-phase system ofclaim 1, where the droplet of a droplet aqueous polymer phase issubstantially spherical.
 7. The aqueous two-phase system of claim 6,where the aqueous two-phase system is in a well with a surface and thesubstantially spherical droplet has minimal contacts with the surface.8. The aqueous two-phase system of claim 1, where the aqueous two-phasesystem is in a well with a surface that is non-adherent to cells.
 9. Theaqueous two-phase system of claim 1, where the aqueous two-phase systemincludes an extracellular matrix material selected from collagen,laminin, fibronectin, matrigel, elastin, and combinations thereof.
 10. Amethod of screening potential drugs comprising: providing the aqueoustwo-phase system of claim 1; administering a potential drug; andmonitoring the three-dimensional aggregate of cells.
 11. The method ofclaim 10, where the three-dimensional aggregate of cells is a cellularspheroid.
 12. The method of claim 11, where a stain is applied to thecells of the cellular spheroid.
 13. The method of claim 11, where theaqueous two-phase system contains a cell culture media.
 14. The methodof claim 13, where the cell culture media is periodically refreshed. 15.The method of claim 14, where the cell culture media is periodicallyrefreshed by automated robotic means.
 16. The method of claim 11, werethe where the aqueous two-phase system is in a well, which is one of aplurality of wells on a well plate.